1. Field of the Invention
The present invention relates to image guided protocol for cell generation. More particularly, the present invention relates to computerized image guided methods for determining and controlling readiness, recovery and rejection of steps in cell generation process.
2. Description of the Related Art
The advancement in stem cell technologies enable a new patient-specific or “personalized” medicine paradigm in 1) autologous regenerative medicine; 2) disease cell lines from patient cells for more predictive assays in drug discovery and basic research; and 3) patient-specific cell generation for personalized diagnostic and drug efficacy or adverse effects testing.
The reprogramming (dedifferentiation) of matured cells such as somatic cells to an embryonic-like state, “induced pluripotent stem cells” (iPSCs), offers the prospect of capturing cells derived from a large number of specific types of pre-diagnosed adult patients, potentially at any age, and a correspondingly large number of controls in a format that can support an industrial level of screening, efficacy, and safety studies. However, to achieve the promise of iPSC technology requires the controlled differentiation of iPSCs to specific cell lineages.
An alternative way of producing differentiated cells is by direct reprogramming or called transdifferentiation which is based on prior identification of transcription factors important in lineage specification. For example, it was shown that pancreatic exocrine cells could be converted in vivo to pancreatic β-cells by infecting them with adenovirus expressing three transcription factors, Ngn3, Pdx1 and Mafa, all known to be important for β-cell development. Similarly, direct reprogramming was shown to convert fibroblasts into neurons and cardiac myocytes.
Another possibility is based on partial dedifferentiation with a subsequent differentiation step. Under certain circumstances, this method might allow for sufficient and rapid expansion of a type of progenitor cell still capable of multilineage differentiation.
The prior art implementation of the cell generation (differentiation) protocols uses a stepwise differentiation method. It regulates cell generation through sequential stages of differentiation. FIG. 1 shows an ideal stepwise cell generation processing flow. The source cell 100 could be iPSCs, partial dedifferentiation cells, embryonic stem cells or matured cell types such as fibroblasts or bloods. In the generation steps 102, 108, 118 certain factors 104, 110, 116 such as Ngn3, Pdx1 are added and the cell generation process progresses to intermediate states 106, 112, 114. The intermediate states in a β-cell generation protocol could include definitive endoderm, endocrine progenitor, pancreatic progenitor, etc. Note that some direct reprogramming protocols bypass progenitor cell state. But for a general framework, we broadly call the intermediate states as different progenitor cells 106, 112, 114. The generation steps 102, 108, etc. continue until reach the final step (corresponding to generation step K 118 in FIG. 1) and this results in the generation of the target cell 120.
The stepwise approach mimics embryonic development process occurring as a series of generation steps, with cells that have multipotential capacity becoming increasingly differentiated. Protocols producing cardiac myocytes from ESCs and iPSCs by sequentially adding morphogenic factors important in the appearance of cardiac muscle are common now. A typical protocol has predefined generation steps. The duration and the timing and amount for the addition of factors at each generation step are also predefined. However, the yield of the protocols is low. This is partially due to many challenges in the complex environment around the cell generation. Key challenges for a cell generation protocol include                efficient induction of the correct germ layer;        correct timing of activation or inhibition of various morphogenic pathways, especially given that the very same pathway can have a stimulatory or an inhibitory influence at different times;        careful control of the concentration of the inducing factors.        
Quantitative markers at different stages of development could be used to assess the results of the intermediate stages. As shown in FIG. 2. Marker set 1 200 is used in probe 1 202 to assess progenitor 1 106 for the step 1 confirmation 204. Similarly, Marker set K-1 206 is used in probe K-1 212 to assess progenitor K-1 114 for the step K-1 confirmation 210.
However, the markers themselves exhibit a great deal of variability because there are significant variations among different cell lines, perhaps because individual lines may make variable amounts of their own inducing factors. In addition, markers introduce additional cost and could have adverse side-effect such as toxicity to the cell generation protocol. Therefore, quantification markers are not desirable in practical clinical laboratory settings. Markers are often used only during the protocol development stage to figure out the proper generation step, duration and timing. During the implementation of the protocol, only a limited number of simple markers could be used, especially for the mass production of patient-specific cells. It is desirable to quantify using non-invasive imaging modalities such as phase contrast images without markers that often require florescence imaging. The low yield and the lack of adequate feedback during the protocol implementation represent a significant hurdle of production level cell generation.